Assays for diagnosis of thrombophilic disease

ABSTRACT

The present invention provides methods for diagnosing and/or monitoring thrombophilic disease in a patient that can result from the antiphospholipid antibody syndrome (aPL syndrome). The methods of the invention are premised on the inhibition of binding of an anticoagulant protein, annexin, preferably annexin-V, to phospholipids by antiphospholipid (aPL) antibodies in a patient blood sample.

The present application is a claim benefit of U.S. applicationProvisional Ser. No. 60/052,313 filed Jul. 11, 1997, the disclosure ofwhich is specifically incorporated herein by reference.

The invention disclosed herein was carried out with grants from the U.S.government. The U.S. has certain rights to the invention.

SPECIFICATION BACKGROUND OF THE INVENTION

The present invention is directed to assays for detection of athrombophilic disease in a patient. The assays, which are based on thediagnosis of the antiphospholipid antibody syndrome (aPL syndrome) in anindividual, detect the capacity of a patient sample to inhibit thebinding of annexin-V, an anticoagulant protein, to a phospholipidsubstrate, and thereby block the anticoagulant activity of annexin-V.

The presence of antibodies in blood which recognize anionicphospholipids, or anionic phospholipid protein complexes, has beenassociated with a thrombophilic syndrome known as the antiphospholipid(aPL) antibody syndrome. The aPL syndrome, which is also known as thelupus anticoagulant syndrome, or the anticardiolipin antibody syndrome,is characterized by arterial and venous thrombosis or, by recurrentpregnancy loss attributed to placental thrombosis (Lockwood C. J., RandJ. H., 1994, Obstet. Gyn. Survey 49: 432; Lockshin M. D., 1996, Lupus 5:404; Shapiro S. S., 1996, Annu. Rev. Med. 47: 533; Asherson R. A,Khamashta M. A., Ordi Ros J., Derksen R. H. W. M., Machin S. J.,Barquinero J., Outt H. H., Harris E. N., Vilardell-Torres M., Hughes G.R. V., 1989, Medicine 68: 366). The aPL syndrome thus comprises athrombophilic disease distinguished by antibodies that recognize anionicphospholipid complexes. The disorder may occur spontaneously or inconjunction with another autoimmune disorder such as systemic lupuserythematosus (Conley C. L., Hartmann R. C., 1952, J. Clin. Invest. 31:621), hence the name “lupus anticoagulant.”

Paradoxically, aPL syndrome antibodies appear to manifest as “lupusanticoagulants” (Conley et al. 1952, J. Clin. Invest. 31: 621; ShapiroS. S., 1996, Annu. Rev. Med. 47: 533; Triplett D. A., 1996 Lupus 5: 43)in vitro by inhibiting phospholipid-dependent blood coagulation. Yet, invivo, these “anticoagulants” are associated with thrombotic mechanisms,and rarely, if ever, with any bleeding disorders.

Evidence has been accumulating that the aPL syndrome bears relationshipto the presence of anticoagulant proteins found on the surface ofendothelial cells that come into contact with blood. Such anticoagulantproteins include a family of proteins known as annexins, a principalmember being annexin-V. Annexin-V, which is also known as placentalanticoagulant protein—1 or vascular anticoagulant—α, has potentanticoagulant properties in vitro that are based on its high affinityfor anionic phospholipids and its capacity to displace coagulationfactors from phospholipid bilayers (Andree et al., 1992, in Andree (ed.)Maastricht, the Netherland, Universitaire pers Maastricht, p. 73).Annexin-V, which is normally present on the apical surface of placentalsyncytiotrophoblasts, has been found to be reduced on apical membranesof placental villi from aPL syndrome patients (Krikun et al., 1994,Placenta 15: 601; Sammaritano et al., 1992, J. Clin. Immunol. 12: 27).

The aPL syndrome has been shown to be an important risk factor forstroke, equivalent in predictive value to hypertension (Rand, J H, 1998,Am. J. Med. Sci. (In press)). Moreover, aPL antibodies are associatedwith recurrent arterial and venous thrombosis. Approximately 40% ofpatients on hemodialysis have aPL antibodies, which may be associatedwith graft thrombosis (the problem of graft thrombosis alone has anestimated cost in the United States of $700 million/year, which is equalto the cost of hemodialysis). aPL antibodies have also been associatedwith recurrent pregnancy loss, however, current diagnostic methods havenot proven specific enough to allow for treatment before a woman hasexperienced at least three spontaneous abortions. Currently, it isrecommended that patients diagnosed for aPL antibodies with thrombosisreceive high intensity anticoagulant therapy for the remainder of theirlives. Thus there is a need for developement of specific assays fordiagnosis and monitoring of thrombophilic disease in susceptibleindividuals, i.e., those known to have aPL syndrome or at risk for aPLsyndrome.

Such methods can be useful for screening for risk of stroke, recurrentmiscarriages and risk of thrombosis or embolism, as well as evaluatingfor the etiology of patients presenting with the above disorders, all ofwhich may be classified as thrombophilic diseases. In addition, therehas been an increasingly frequent clinical problem in the identificationof patients with incidental positive antiphospholipid antibody tests(7-10% of the general population, and up to 40% of ill hospitalpatients) with no accepted means to distinguish abnormalities that areclinically relevant (disease related) from those that are irrelevant.The present invention is directed to providing specific assays to beused as a basis for making those distinctions.

The methods of the present invention are based upon the interaction ofphospholipid substrates, aPL antibodies and annexin-V and, also, theconsequent coagulant activity of the complex exposed to clottingfactors. The methods can be used to diagnose thrombophilic disease (orhypercoaguable disease) in susceptible individuals (i.e., those havingor at risk of developing aPL syndrome) by utilizing the reduction inbinding of annexins, particularly annexin-V, to phospholipid substratesin the presence of a blood specimen from a known or suspected aPLsyndrome patient as a marker of the disease. The present invention thusprovides for specific assays which are predictive of and can monitorthrombophilic disease, as well as the success of treatment.

SUMMARY OF THE INVENTION

The present invention provides methods for diagnosing and/or monitoringthrombophilic disease in a patient that can result from theantiphospholipid antibody syndrome (aPL syndrome). The methods of theinvention are premised on the inhibition of binding of an anticoagulantprotein, annexin, preferably annexin-V, to phospholipids byantiphospholipid (aPL) antibodies in a patient blood sample.

in one aspect, the method involves incubating a phospholipid substratewith a test blood specimen (plasma, serum, isolated IgG) in the presenceof a known amount of an annexin, preferably annexin-V, for a timesufficient to allow the annexin to bind to the phospholipid substrate.The unbound annexin and specimen are removed from the substrate and theamount of annexin bound to the substrate in the presence of the testspecimen is measured and compared to an amount of annexin bound in thepresence of a control specimen (known to not contain aPL antibodies),wherein a lower amount of annexin bound in the presence of the testspecimen versus the control indicates the presence of thrombophilicdisease in the individual.

In a variation of the above method, the amount of unbound annexin ismeasured, wherein a higher amount of unbound annexin in the testspecimen compared to the control indicates thrombophilic disease in thepatient.

In a further aspect of the invention, the method involves diagnosingand/or monitoring thrombophilic disease in a patient using a coagulationassay. The latter methods are premised on the anticoagulant effect ofannexin (annexin-V), which effect is reduced or inhibited by aPLantibodies. The coagulation assay may be carried out as a one or twostage assay.

In a one stage assay, anticoagulated patient plasma is incubated induplicate with a phospholipid dependent coagulation test reagent,followed by calcifying the plasma to induce clotting. In one of theduplicate samples, annexin is added and in the other, annexin is notpresent. The time to clot formation in both samples, wherein a reducedanticoagulant effect in the presence of annexin indicates thrombophilicdisease.

In a two stage assay, anticoagulated patient plasma, serum or IgG isincubated in duplicate with the coagulation test reagent. The patientsample is removed and the duplicate reagents are incubated further witha sample of control plasma, preferably pooled normal plasma. The samplesare then calcified in the presence and absence of annexin, as above, andmonitored for time to clot formation. Again, a reduced anticoagulationeffect in the presence of annexin indicates thrombophilic disease.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C. Effects of Antiphospholipid-Antibody IgG on Annexin-V andPlasma Coagulation on Trophoblasts. Cultured trophoblasts (from the BeWocell line) grown to confluence were exposed to IgG preparations (2 mgper milliliter) from three patients and their controls for two hours at4° C. to inhibit the recycling of membranes and vesicles. Annexin-V wasthen dissociated with buffer containing ethylene glycol-bis(β-aminoethylether)N,N,N′,N′-tetraacetic acid EGTA) and measured by immunoassay. (Alltests were performed in quadruplicate.)

FIG. 1A shows that the mean (±SE) level of annexin V, indicated by thehorizontal line and error bar, was significantly lower after exposure toantiphospholipid IgG than after exposure to control IgG (0.37±0.02 vs.0.85±0.12 ng per well, P=0.02).

FIG. 1B shows how antiphospholipid IgG affects annexin-V levels onprimary cultured trophoblasts and BeWo trophoblasts. (The data on theformer were normalized for the DNA concentration, and both sets of datawere normalized as percentages of the control values so that the twocell types could be shown together.) Annexin-V levels on the surface ofboth types of trophoblasts were significantly reduced (P<0.001 forboth).

FIG. 1C shows the coagulation time of plasma added to BeWo trophoblastsexposed to preparations of IgG from the three patients for two hours at4° C., as compared with controls. In these experiments, annexin-V wasnot dissociated from the cells. The mean (±SE) coagulation time wassignificantly shorter in the antiphospholipidIgG-exposed trophoblaststhan in the controls (8.7±2.0 vs. 21.3±2.9 minutes, P=0.02).

FIGS. 2A-D. Effects of Antiphospholipid-Antibody IgG on Annexin-V andPlasma Coagulation on Umbilical-Vein Endothelial Cells. Umbilical-veinendothelial cells were exposed to IgG preparations from three patientsand their controls for two hours at 4° C. to inhibit the recycling ofmembranes and vesicles.

FIG. 2A shows that the mean (±SE) level of annexin V, indicated by thehorizontal line and error bar, was significantly lower after exposure toantiphospholipid IgG than after exposure to control IgG (1.6±0.04 vs.2.1±0.05 ng per well, P=0.001).

FIG. 2B shows the coagulation time of plasma added to these cultures ofendothelial cells. Overall, the mean (±SE) coagulation time wassignificantly shorter in the three groups ofantiphospholipid-antibody-exposed endothelial cells than in the controls(9.8±0.8 vs. 14.2±1.2 minutes, P=0.04).

FIG. 2C shows the annexin-V levels after endothelial cells were culturedwith preparations of antiphospholipid IgG from the three patients andcontrol IgG for 20 hours at 37° C., a temperature at which recycling ofmembranes and vesicles occurs. The mean (±SE) level of annexin-V in thecells exposed to antiphospholipid IgG was significantly lower than thelevel in the control cells (1.3±10.2 vs. 2.1±0.1 ng per well, P=0.02).

FIG. 2D shows the coagulation time of plasma added to endothelial cellscultured with IgG preparations from the three patients and the controlsfor 20 hours at 37° C. Again, the coagulation time was significantlyshorter for the endothelial cells exposed to antiphospholipid antibody,with a lower mean value in those cells than in the control cells(17.2±0.2 vs. 23.5±1.2 minutes, P=0.006).

FIGS. 3A-B. Effects of Polyclonal Antiannexin Antibodies and PurifiedAnnexin-V on the Coagulation of Plasma Exposed to Umbilical VeinEndothelial Cells.

FIG. 3A shows that the mean (±SE) coagulation time of plasma wassignificantly less after cells were incubated with rabbit polyclonalanti-annexin-V as compared with equal concentrations of control rabbitpolyclonal IgG (20.1±0.6 vs. 23.8±0.5 minutes, P=0.006). Treatment withrabbit polyclonal anti-annexin II had no effect (coagulation time,23.8±0.8). When cells were pretreated with EGTA, which dissociatescell-surface annexin V, there was no difference between the resultsobtained with the various antibodies.

FIG. 3B shows how dissociating and restoring annexin-V affects thecoagulation of plasma exposed to umbilical-vein endothelial cells. Theplasma coagulation time was significantly shorter after annexin-V wasdissociated from the cell surface by treatment with EGTA alone ascompared with calcium (mean of eight experiments, 14.6±0.9 vs. 22.5±0.5minutes; P<0.001). When exogenous annexin-V was added to the culture,dose-dependent prolongations of plasma coagulation were observed. Thedifference in the coagulation time between cells treated withethylenediaminetetraacetic acid (EGTA) and those treated with calciumwas also significant when 1 μg of annexin-V was added per milliliter(P<0.001).

FIGS. 4A-D. Model for mechanisms of the “lupus anticoagulant effect” andthe inhibition of annexin-V and acceleration of coagulation byantiphospholipid antibodies.

FIG. 4A Anionic phospholipids (negative charges), when exposed on theapical surface of the cell membrane bilayer, serve as potent cofactorsfor the assembly of three different coagulation complexes: the tissuefactor (TF)-VIIa complex, the IXa-VIIIa complex and the Xa-IXa complex,and thereby accelerating blood coagulation. The TF complexes yieldeither factor Va or factor Xa, the IXa complex yields factor Xa(prothrombinase), and the Xa formed from both of these reactions is theactive enzyme in the prothrombinase complex which yields factor Ia(thrombin), which in turn cleaves fibrinogen to form fibrin.

FIG. 4B Annexin-V, in the absence of aPL antibodies, forms clusterswhich bind with high affinity to the anionic phospholipid surface andshield the surface from the assembly of the phospholipid-dependentcoagulation complexes, thereby inhibiting coagulation reactions.

FIG. 4C In the absence of annexin-V, aPL antibodies can prolong thecoagulation times, compared to control antibodies, by reducing theaccess of coagulation factors to anionic phospholipids. This may resultin a “lupus anticoagulant” effect.

FIG. 4D However, in the presence of annexin-V, antiphospholipidantibodies, either directly or via interactior with protein-phospholipidcofactors, disrupt the the ability of annexin-V to cluster on thephospholipid surface, resulting in a net increase of the amount ofanionic phospholipid to available for promoting coagulation reactions.This manifests in the net acceleration of coagulation in vitro and inthrombophilia in vivo.

FIGS. 5A-D. Ellipsometry studies of effects of aPL IgG and cofactor ondisplacement of annexin-V from PS/PC phospholipid bilayers.

FIG. 5A shows the rapid adsorption of annexin-V to the PS/PC (30%/70%)phosphoilpid bilayer. Treatment with EDTA and measurement of thedesorption of this protein can be used to measure the amount ofannexin-V on the phospholipid surface. As shown, this calcium-dependentbinding protein is completely desorbed from the phospholipid surface byaddition of 6 mM EDTA,

FIG. 5B shows that incubation of the annexin-V coated phospholipidbilayer with a polyclonal human aPL IgG in the absence ofβ₂-glycoprotein I does not displace the annexin-V—i.e. the quantity ofannexin-V desorbed after treatment With EDTA matches the quantity ofannexin-V which had originally adsorbed,

FIG. 5C shows incubation of the annexin-V coated phospholipid bilayerwith β₂-GP I followed by polyclonal aPL IgG results in a significantreduction of the quantity of annexin-V on the bilayer. This is reflectedby the marked reduction of the amount of annexin-V which desorbs aftertreatment with EDTA.

FIG. 5D shows treatment of the phospholipid bilayer with the β₂-GP Icofactor followed by a control (non-aPL) IgG) fraction does not changethe quantity of annexin-V on the phospholipid surface at all—i.e., thequantity of annexin-V which is desorbed by treatment with EDTA is thesame as the quantity which had been adsorbed in the first place.

FIG. 6 shows quantitative displacement of annexin-V from the PS/PCphospholipid bilayers by aPL IgG preparations in the presence of β₂-GP Icofactor. The combination of aPL IgG with β₂-GP I significantly displaceannexin-V from the bilayers as compared to control IgG with β₂-GP I. Themean (±SEM) quantity of annexin-V displaced by 3 different aPL syndromepatients' IgG fractions was 0.115±0.014 lg/cm² as compared to nosignificant displacement by 3 different control IgG preparations(0.005±0.007 μg/cm², p=0.002). These data demonstrate the displacementof annexin-V from the phospholipid bilayer surface by aPL IgG in thepresence of β₂-GP I.

FIG. 7 shows annexin-V binding to microtiter plates coated withphosphatidyl serine (PS). PS-coated microtiter plate wells treated withaPL plasmas bound significantly less biotin-labeled annexin-V thanPS-coated microtiter plate wells which had been treated with controlplasmas. Annexin-V was detected by addition of phosphatase-labeledstreptavidin followed by p-nitrophenyl phosphate substrate. The mean OD(±SEM) of the aPL plasma-treated wells was 0.085±0.003 and was0.123±0.005 for the wells treated with control plasmas (n=10 for eachgroup, p<0.0001).

FIGS. 8A-B. The effects of aPL IgG on the quantity of platelet surfaceannexin-V and plasma coagulation.

FIG. 8A. Washed human platelets were exposed to 3 different aPL andcontrol IgG preparations in plasma, following which the platelets wereincubated with annexin-V (20 μg/ml), in the presence of calcium, asdescribed in Methods. Surface annexin-V was then dissociated with EGTAand measured by Enzyme Linked Immunosorbent Assay (ELISA). Plateletspreexposed to aPL IgG had significantly less annexin-V on their surfaces(mean±SEM-0.89±0.12 ng/l 0⁶ platelets) as compared to controls(2.01±0.38 ng/l 0⁶platelets, p=0.05).

FIG. 8B. Plasma coagulation times were determined using platelets whichhad been pre-exposed to the aPL and control IgGs in plasma. Theplatelets were added to pooled normal plasma which was recalcified inthe presence and absence of added annexin-V (20 μg/ml). Annexin-Vlengthened the coagulation times of pooled normal plasma with bothcontrol and aPL IgG-treated platelets. However, the net prolongation,compared to the coagulation time without annexin-V, was significantlyless with the aPL-treated platelets (mean prolongation±SEM: 33.2±0.9sec) as compared to controls (50.4±4.1 sec, n=3, p=0.01).

FIGS. 9A-B. The effects of aPL plasmas on annexin-V bound to aPTTreagent-phospholipid and plasma coagulation with this reagent.

FIG. 9A. aPTT reagent-phospholipid was exposed to 4 different aPL andcontrol plasmas and then to annexin-V (2 μg/ml), after which surfaceannexin-V was dissociated with EDTA and measured by ELISA. aPTTreagent-phospholipid which had been pre-exposed to aPL-piasmas hadsignificantly less annexin-V (mean±SEM: 318±28 ng/50 μl aliquot ofreagent) as compared to controls (656±80 ng/50 μl aliquot of reagent,p=0.01).

FIG. 9B. Plasma coagulation times were determined using aPTTreagentphospholipid exposed to the aPL and control plasmas (n=10 foreach group) in the first stage and then in the second stage, to poolednormal plasma in the presence and absence of added annexin-V (30 μg/ml).Annexin-V delayed the coagulation times of aPTT reagent exposed to bothtypes of plasmas. In the presence of annexin-V, the coagulation times ofthe aPTT reagent which had been pre-exposed to aPL-plasma wassignificantly faster (mean±SEM: 89.2±9.2 sec) than reagent exposed tothe control plasma (102.5±2.6 sec, p=0.001). Also, there was asignificant decrease in the net prolongation of the coagulation timesinduced by annexin-V (mean±SEM: 13.6±1.8 see for aPL plasmas versus23.1±0.8 sec for controls, p=0.0002).

FIGS. 10A-B. The effects of aPL plasmas on annexin-V bound toprothrombin time reagent (tissue factor-phospholipid complex) and onplasma coagulation.

FIG. 10A. Prothrombin time reagent was pre-exposed to 3 different aPLand control IgG preparations in plasma and then to annexin-V (2 μg/ml),after which surface annexin-V was dissociated with EDTA and measured byELISA. Prothrombin time reagent pre-exposed to aPL IgG-containingplasmas had significantly less annexin-V (mean±SEM−82±4 ng/50 μl aliquotof reagent ) compared to controls (110±1 ng/50 μl aliquot of reagent,p=0.02).

FIG. 10B. Plasma coagulation times were determined using prothrombintime reagent which was exposed to the aPL and control plasmas (n=10 foreach group) in the first stage and, in the second stage, to poolednormal plasma in the presence and absence of annexin-V (30 μg/ml). Therewas a small but significant prolongation of coagulation time when theprothrombin time reagent was exposed to the aPL plasmas in the absenceof annexin-V (p=0.003). Addition of annexin-V resulted in prolongationof coagulation times with both types of reagent (i. e. exposure tocontrol and aPL plasmas). However, in contrast to the results withoutannexin-V, the coagulation times of the prothrombin time reagent whichhad been pre-exposed to aPL plasma were significantly shortened(mean±SEM: 35.0±0.8 sec compared to 38.3±1.2 sec for control plasmas,p=0.03). There was a concomitant significant decrease in the netprolongation of the coagulation times using PT reagent which had beenpretreated with aPL plasma (10.3±0.8 sec compared to 15.2±1.2 sec forcontrol plasmas, p=0.004).

FIG. 11 shows the effects of aPL plasmas on the binding ofFITC-annexin-V to aPTT reagentphospholipid, aPTT reagent was incubatedwith aPL and control plasmas (n=10 for each group), after which 1 μg/mlof FITC-annexin-V was added. Annexin-V bound to the aPTT reagent and inthe fluid phase were quantified by spectrofluorimetry, as described inMethods. There was a significant decrease of the amount of annexin-Vassociated with the aPTT reagent which had been pre-incubated with aPLplasmas (mean±SEM−0.083±0.008 RFU/50 μl aliquot of reagent) compared tocontrols plasmas (0.131±0.015 RFU/50 μl aliquot of reagent, p=0,01). Incontrast, there was a significant increase in the amount of labeledannexin-V remaining in the supernatant of aPTT reagent which had beenpreincubated with aPL plasmas (mean±SEM: 0.070±0.008 RFU/50 μl aliquotof reagent) as compared to control plasmas (0.046±0.005 RFU/50 μlaliquot of reagent, p=0.02).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of detecting and/ordiagnosing thrombophilic disease in an individual that can result fromthe antiphospholipid antibody syndrome (aPL syndrome). The methods ofthe invention involve measuring the inhibition of annexin-V binding toanionic phospholipid substrates by antiphospholipid (aPL) antibodies ina patient blood sample. The methods are particularly useful indiagnosing and/or monitoring thrombophilic disease in susceptibleindividuals, i.e., those known to have or be at risk for the aPLsyndrome. Such individuals undergo accelerated coagulation of theirblood compared to normal individuals, and are at increased risk tosuffer arterial and/or venous thrombosis and spontaneous pregnancylosses (i.e. miscarriages). The increased risk of stroke, recurrentmiscarriages, and risk of thrombosis or embolisms correlated with theaPL syndrome may all be classified as thrombophilic (or hypercoagulable)diseases.

The present invention is premised on the finding, disclosed herein, of anew thrombogenic mechanism in the aPL syndrome—that IgG fractions fromaPL patients reduce the quantity of annexin-V on cultured trophoblastsand endothelial cells and accelerate the coagulation of plasma added tothese cells. In addition, the present invention also provides that thiseffect also occurs with other phospholipid substrates, such asphospholipid-coated surfaces, frozen thawed washed platelets andphospholipid suspensions used for conventional clinical coagulationtests. Thus, the phospholipid substrates useful in the present inventioncan include, inter alia, cultured trophoblasts, endothelial cells(particularly human umbilical vein endothelial cells—HUVECs), othercells and cell lines that display surface anionic phospholipids,platelets, phospholipid coated silicon wafers, phospholipid coatedmicrotiter plates, phospholipid coated beads, phospholipid suspensionsand clinical coagulation test reagents comprising phospholipids. Thephospholipids include, inter alia, phosphatidyl serine, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin,phosphatidic acid and combinations thereof. Preferred phospholipidsinclude phosphatidyl serine, phosphatidyl choline and combinationsthereof.

In accordance with the invention, ellipsometry studies have provideddirect evidence that aPL IgG preparations, in the presence of acofactor, e.g., β₂-glycoprotein I (β₂-GP I), displace annexin-V fromphosphotidyl serine/phosphatidyl choline (PS/PC) phospholipid bilayers.β₂-GP I is a serum factor that appears to be required for aPL antibodybinding to phospholipids (Roubey 1994, Blood 84:2854, incorporatedherein by reference). In addition, other proteins, such as prothrombin,may also serve as cofactors. In accordance with the invention, when aPLIgG is used in practice, both the aPL IgG and β₂-GP I are believednecessary for annexin displacement to occur. β₂-GP I is not required tobe added if aPL plasma or serum is used. It has also been found thatincubating PS-coated microtiter plate wells with aPL patient plasmasresults in the significant reduction of the quantity of labeledannexin-V which binds to this phospholipid substrate.

In addition, exposing phospholipid substrates used for coagulationreactions, such as washed frozen thawed platelet suspensions, partialthromboplastin time (aPTT) reagent and prothrombin time (PT) reagent, toaPL antibodies reduces the quantity of annexin which binds to thesephospholipid substrates. Using cell cultures, platelets and non-cellularphospholipid substrates, the aPL antibodies also accelerate coagulationin the presence of anticoagulant proteins, such as annexin, reducing thepotent anticoagulant activity of this phospholipid-binding protein.

The novel findings underlying the invention, which demonstrateaccelerated coagulation by aPL antibodies when annexin-V is present incoagulation reactions, stand in contrast to the “lupus anticoagulant”phenomenon which was first described in 1952 (Conley, 1952, J. Clin.Invest. 31:621). The present invention provides the first directdemonstrations of the displacement of annexin-V and the acceleration ofcoagulation on noncellular phospholipid substrates by aPL antibodies andprovide a methodology suitable for clinical testing for thrombophilicdisease. While the invention exemplifies the use of annexin-V in themethods for testing for thrombophilic disease, other members of theannexin family (Tait et al., 1988, Biochemistry 27: 6268, incorporatedherein by reference), as well as other phospholipid-bindinganticoagulant proteins, are encompassed within the scope of theinvention. Since annexin-V has the highest affinity for phospholipidamong the various members of the annexin family, it is believed that aPLantibodies will have a similar effect on displacing lower affinityannexins, e.g. annexin-II, which might also have antithromboticproperties (Hajjar et al., 1996, J. Biol. Chem. 271:21652, incorporatedherein by reference) from phospholipid substrates.

Since, in accordance with the invention, aPL antibodies are capable ofreducing the binding of annexin-V to anionic phospholipids in vitro andin cell culture, it is believed that this effect also occurs in vivowhere anionic phospholipids are exposed to flowing blood. Suchsituations and processes include the surface of placental villi (Lydenet al., 1992, J. Reprod. Immunol. 22:1), activated platelets(Thiagarajan et al., 1990, J. Biol. Chem. 265:17420), apoptic cells andcellular particles (Martin et al., 1995, J. Exptl. Med. 182:1545) anddisrupted erythrocyte membranes. In all of these settings, the presenceof aPL antibodies could inhibit the binding of circulatinganticoagulants, such as annexin-V, which, in turn, results in a membranesurface which is more thrombogenic.

Thus, according to the invention, aPL antibodies have a prothrombiceffect on phospholipid substrates in vivo and in vitro. The mechanism bywhich aPL antibodies accelerate coagulation in the presence of annexin-Vis believed to be due to topographical differences between the bindingof the two ligands (aPL antibodies and annexin) to phospholipidsubstrates. Annexin-V binds in clusters on the phospholipid surfacewhile aPL antibodies disrupt this carpet of annexin-V, which normallywould shield the surface and allow enough room on the phospholipidsurface for coagulation factors such as prothrombin to bind. Thus, theprothrombotic effect of the aPL antibodies is believed to be due totheir increasing the net quantity of available anionic phospholipid forcoagulation reactions by displacing annexin-V from the surface. In viewof the present findings, it is believed that the afore mentionedparadoxical “lupus anticoagulant” phenomenon is a reflection of theeffects of the aPL antibodies in the presence of coagulation proteins inplasma, but in the absence of annexin-V; in this situation theantibodies will prolong coagulation by decreasing the net quantity ofphospholipids available for coagulation reactions (since annexin-V isnot present). In reflecting the relative inhibitory effects of highaffinity antiphospholipid-protein complexes—in the absence ofannexin-V—to the binding of coagulation factors, the invention alsoprovides that the “lupus anticoagulant” phenomenon may indeed serve as asurrogate marker for antibodies whose actual pathophysiogic functionlies in their capacity to displace annexin-V.

In accordance with the present invention, FIG. 4 depicts a modelunderlying the basis for the methods provided herein. Anionicphospholipids on the apical surfaces of cell membranes serve as thecofactor for coagulation complex, which accelerate blood clotting (FIG.4A). Annexin-V is believed to play a physiologic role in inhibitingblood coagulation reactions on vascular surfaces (phospholipidssubstrates) by shielding highly thrombogenic anionic phospholipids fromcoagulation enzyme complexes (FIG. 4B). It is believed that aPL syndromepatients have antibodies with a sufficient affinity for anionicphospholipids (and/or presumptive cofactors, such as β₂-GP I) to disruptor prevent the assembly of this annexin-V protective shield. Thisresults in a net increase of exposed phospholipid and permits a moreprocoagulant topography to be available for coagulation reactions. Thus,coagulation systems which include annexin-V will demonstrate a relativeacceleration of coagulation—a “lupus procoagulant effect”—due todisplacement of annexin-V by aPL syndrome antibodies (FIG. 4D). Incontrast, in the absence of added annexin-V, only the relativelyinhibitory effects of the antibodies on phospholipid-dependentcoagulation reactions are observed (as compared to when antibodies areabsent), thereby resulting in the classical “lupus anticoagulant” effect(FIG. 4C).

With reference to FIG. 4A, according to the present invention, it hasnow been shown that the binding of annexin-V to the phospholipidsubstrate inhibits prothrombinase (factor Xa) activity, therebyinhibiting the formation of thrombin. Annexin-V normally displacesprothrombinase (and also other phospholipid dependent complexes) fromthe phospholipid substrate, thus inhibiting prothrombinase activity, aPLantibodies (isolated IgG and cofactor) interferes with the annexindisplacement of prothrombinase, thereby promoting clotting.

Thus, in one embodiment of the invention, a method for diagnosing and/ormonitoring thrombophilic disease in a patient (ie., a patient with orsuspect of having the aPL syndrome) is provided in which the amount ofannexin bound to a phospholipid substrate in the presence of a bloodspecimen from a patient suspected of having thrombophilic diseaserelated to the aPL syndrome is compared to a reference amount of annexinbound to a companion phospholipid substrate in the presence of a controlblood specimen. The control blood specimen may be obtained from a singlesource or, preferably, may be derived from a pool of normal donors. Thelatter provides a reference range that minimizes variations possiblewith individual samples. Such pools can be characterized andstandardized for repeated use in the assays. They are aliquoted andstored frozen for use, providing a ready source of standardized controlspecimens. Following an incubation period, unbound annexin and bloodspecimens are removed and the amount of annexin bound to thephospholipid substrate is measured. The amounts bound in the presence orabsence of test sample are compared. A lower amount of bound annexinfrom the test specimen indicates the presence of aPL antibodies in thespecimen and the likelihood that the individual has thrombophilicdisease related to the aPL syndrome.

The test and control samples can be whole blood, plasma, serum andisolated IgGs. Plasma is preferred to whole blood, but the plasma mustbe anticoagulated for use in the binding assays. Preferably, theanticoagulated plasma is citrated, however, other anticoagulants knownto those of skill in the art, including EDTA, heparin, and hirudin arealso useful. Likewise, serum and, more preferably, isolated IgG can beused. IgG can be isolated from blood samples by any IgG isolationprocedure known to those of skill in the art.

When isolated IgGs are used in the assay, the addition of a cofactor,such as β₂-glycoprotein I, which enhances aPL antibody binding tophospholipids, is preferred. Other cofactors that can be used includeprothrombin. A cofactor is not required when plasma or serum is used.

Annexins used in the invention can be isolated by known techniques fromplacenta or can be synthesized by recombinant DNA techniques known tothose of skill in the art. In a preferred embodiment, the annexin isannexin-V isolated from placenta or obtained by recombinant techniques.

As used in the invention, the annexins can optionally comprise adetectable label which allows for measurement of annexin in the assays.Such labels include, inter alia, biotin, radioisotopes, such as ¹²⁵I and¹³¹I, fluorochromes, such as fluorescein isothiocyanate (FITC),chromophores and any other such labels, such as chemiluminescent labels,known to those of skill in the art. The use of such labels allows forthe design of specific assays, such as radioimmunoassays usingmonoclonal or polyclonal antibodies that specifically interact with theannexin, especially annexin-V, direct fluorescence or chemiluminescencemeasurements compared to a standard protein curve, and the use ofstreptavidin binding to biotin to specifically measure biotin labeledannexin.

Alternatively, unlabeled annexin, particularly annexin-V, can bedetected and quantitated by means of enzyme-linked immunoassays (ELISA),as is well known in the art, utilizing monoclonal or polyclonalantibodies, specific for annexins, particularly annexin-V.

Measurement of the amount of annexin bound to the phospholipid substratecan be performed in situ, i.e., on the substrate, or on annexin removedfrom the phospholipid substrate, e.g., by desorbing with a calciumchelator such as EDTA or EGTA. The afore mentioned measurement assayscan all be performed in situ or on desorbed annexin.

The phospholipid substrates for use in the invention include, interalia, trophoblasts, such as primary trophoblasts or cultured trophoblastcell lines, e.g., BeWo; endothelial cells, preferably human umbilicalvein endothelial cells (HUVECs); other cells and cell lines havingsurface anionic phospholipids; frozen thawed washed platelets;phospholipid suspensions; phospholipid coated silicon wafers;phospholipid coated microtiter plates; phospholipid coated beads andphospholipid dependent clinical coagulation test reagents, such aspartial thromboplastin time (aPTT), prothrombin (PT) reagents and otherphospholipid dependent coagulation tests known to those of skill in theart. The advantages of the coagulation tests, such as aPTT and PTreagents, and platelets are that they can also be used in furthercoagulation measurements as described below.

Phospholipids for use in the invention are known to those in the art andinclude, inter alia, phosphatidyl serine, phosphatidyl choline,phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin,phosphatidic acid and combinations thereof. Preferred phospholipidsinclude phosphatidyl serine, phosphatidyl choline and combinationsthereof.

In another embodiment of the present invention, a similar approach istaken where the annexin is bound to the phospholipid substrate in thepresence or absence of a test blood specimen as described above.However, instead of measuring the amount of annexin bound to thephospholipid substrate, the amount of annexin remaining in thesupernatant is measured, as described above. In this embodiment, ahigher amount of annexin, detected and measured as described above inthe supernatant of test samples compared to controls indicates thepresence of aPL antibodies in the sample which is correlated withthrombophilic disease in the patient.

In a further embodiment of the invention, the methods for detectingan/or monitoring thrombophilic disease utilize measuring coagulation ofplasma in the presence of annexin on phospholipid substrates to detectthe functional consequences of the reduction of annexin, preferrablyannexin-V, by aPL antibodies in a patient sample. The patient sample ispreferably anticoagulated plasma, i.e., plasma in which a knownanticoagulant is present, or, in some cases, IgG. Citrated plasma ispreferably used, although other anticoagulants known in the art, such asEDTA, heparin and hirudin, can be used as well. For such assays, thephospholipid substrate comprises a phospholipid dependent coagulanttest, preferably aPTT or PT reagent or, alternatively, frozen thawedwashed platelets. In such assays, the tests are performed in thepresence and absence of annexin in order to measure the anticoagulanteffect of the protein.

Coagulation assays may be a single stage or two state assay. For singlestage assays, useful for screening a number of patients, anticoagulatedplasma from a patient is incubated with a phospholipid-dependentcoagulation test, such as aPTT or PT reagent. The plasma is thenrecalcified (e.g., using calcium ions, such as CaCl₂, to counteract theeffects of the anticoagulant) in the presence and absence of a knownreference amount of annexin (annexin-V) that provides a standardizedanticoagulant effect, incubated and the time to clot formation ismonitored. In a one stage assay, the patient plasma provides both aPLantibodies and the coagulation factors necessary for clothing. Parallelassays may optionally be carried out with control plasma. A reducedanticoagulant effect seen with the patient plasma in the presence ofannexin indicates thrombolytic disease in the patient.

The coagulation assay may also be carried out as a two stage assay,which is preferred for monitoring individual patients because of greaterconsistency of the assay. In the two stage assay, anticoagulated plasma,serum or IgG, from a patient is incubated with the coagulation testreagent, as above. The patient sample is removed and anticoagulatedpooled normal plasma is added to the test reagent. The latter plasma isthen recalcified, as above, in the presence and absence of a referenceamount of annexin and the time to clot formation monitored. The use ofthe pooled plasma in the second stage minimize the variable baselinecoagulation times of individuals.

In practice of the two stage assay, an aPTT or PT reagent is incubatedwith either anticoagulated patient plasma (or IgG) or control plasma (orIgG), followed by sedimenting the mixture, removing the unbound plasma(or IgG) and resuspending the reagent. Anticoagulated pooled normalplasma is added and, following a brief incubation, a calcium ionsolution (e.g. CaCl₂) with and without annexin (preferably annexin-V) isadded to the samples, whereafter the time to clot formation ismonitored.

As above, for the single stage, if aPL antibodies are present in thepatient plasma, significantly accelerated coagulation times andreductions of the anticoagulant effects of the annexin will be observedwhen compared to control plasma, indicating thrombophilic disease in thepatient. The reduced anticoagulant effects of the annexin is manifest inpatient samples when plasma containing annexin is compared with plasmalacking annexin.

In a still further aspect of the present invention, the methodsdisclosed herein may be useful in detecting low affinity mutant orpolymorphic annexins in patients. Thus, the methods are useful fordetecting annexin in patients that have functional defects such asdecreased anticoagulant and antithrombotic properties.

As disclosed, the present invention provides methods for detecting theability of aPL antibodies in patient plasma to displace annexins fromphospholipid substrates. Functionally defective polymorphic or mutantannexins may also be measured in these assays.

Polymorphic annexins may be isolated by affinity chromatography usingmonoclonal or polyclonal antibodies specific to various annexins, e.g.,annexin-V. The binding of such annexins to phospholipid substrates inthe presence of aPL antibodies can be measured as above. Mutant annexinswith decreased binding are believed to be associated withhypercoagulability, which those with increased binding are believed tobe associated with hypocoagulability (bleeding disorders).

Likewise, the effects of mutant annexins can be measured in thecoagulation assays described herein, using the PT and aPTT reagents.Mutant annexins having decreased anticoagulant effectiveness areassociated with hypercoagulability, while those with increasedanticoagulant effectiveness are associated with hypocoagulability.

Finally in a specific aspect of this embodiment, the effects of purifiedmonoclonal aPL antibodies having low and high affinities on mutantannexin binding to phospholipid substrates and their anticoagulanteffectiveness can be measured. Mutant annexins having decreasedanticoagulant effectiveness are expected to be inhibited by low affinityaPL antibodies and be associated with hypercoagulability. On the otherhand, mutant annexins having increased anticoagulant effectiveness areexpected to resist inhibition by high affinity aPL antibodies and beassociated with hypocoagulability (i.e., bleeding disorders).

EXAMPLE 1

Isolation of IgG

IgG antibodies were isolated from the citrated plasma of three patientswith severe antiphospholipid (aPL)-antibody syndrome and three normalcontrol subjects using a protein G column, as described by Sammaritanoet al., 1992, J. Clin. Immunol. 12:27, incorporated herein by reference.A preparation of antiphospholipid antibody from each of the threepatients was studied and compared with a preparation from one of thecontrols. The three patients all had severe primary aPL-antibodysyndrome, that is, there was no evidence of systemic lupus erythematosusor any other autoimmune disorder, and high titers of anticardiolipinIgG.

The first patient was a 33-year old woman (previously described byOmstein et al., 1994, J. Rheumatol. 21:1360, incorporated herein byreference) who had evidence of a previous cerebral infarct on a computedtomographic scan, previous cerebral infarct on a computed tomographicscan, previous deep-vein thrombosis and pulmonary embolism, and fourconsecutive losses of pregnancy. She presented with a fifth pregnancyloss at 18 weeks' gestation, placental infarction, and infarcts on theskin of her hands and face, with fibrin thrombi in the small vessels ofthe dermis. The second patient was a 47 year-old man with catastrophicantiphospholipid syndrome, manifested by deep-vein thrombosis, pulmonaryemboli, and stroke. The third patient was a 63-yearold woman withstroke, pulmonary embolism, and infarcts on the skin of her hands.

EXAMPLE 2

Plasmas

For studies with plasmas, citrated specimens were collected from 10patients with aPL syndrome and 10 non-aPL controls at the Mount SinaiMedical Center, New York, N.Y. The IgGs and plasmas were tested for thepresence of antibodies against β₂-GP I, prothrombin and annexin-V withstandardized nitrocellulose (Schleicher & Schueli, Keene, NH) dot-blotscontaining varying quantities of the proteins, up to 1 μg. All 3 of thepurified aPL IgGs recognized β₂-GP I directly, one of the 3 recognizedpurified human prothrombin, and none recognized annexin-V directly. Ofthe 10 aPL plasmas, nine contained immunoglobulin which recognized β₂-GPI, one recognized prothrombin alone, and none recognized annexin-Vdirectly. To provide the same standard plasma for the second stage ofcoagulation tests, plasmas from three normal blood bank donors werepooled, alliquotted and stored at −40° C. for all coagulation studies.

EXAMPLE 3

Annexin-V

Annexin-V was purified from human placentas as described by Yoshizaki,et al., 1989, J. Biochem. (Tokyo) 105:178, incorporated herein byreference. The identity of the protein was confirmed by immunoblotanalysis using a previously characterized affinity purified monospecificpolyclonal rabbit anti-annexin-V IgG (Krikun et al., 1994, Placenta15:601, incorporated herein by reference). For studies of annexin-Vbinding to phosphatidyl serine-coated microtiter plates (describedbelow), the protein was labeled with biotin as previously described(Flaherty et al., 1990, J. Lab. Clin. Med. 115:174, incorporated hereinby reference). Annexin-V was dialyzed at 4° C. against buffer containing0.05 M boric acid, 0.1 M NaCl, pH 8.5. Biotin-NHS (CalbiochemNovabiochemCorporation, La Jolla, Calif.) was then added to the annexin-V at a 1:2molar ratio of annexin-V to biotin-NHS. The reaction was carried out for30 min at 4° C. and quenched with 10 mM glycine. The biotinylatedannexin-V was then dialyzed against TBS buffer (0.05M Tris, OAM NaCl, pH7.4). The concentration of biotinylated annexinV was determined byabsorbance at 280 nM and aliquots were stored at −70° C.

EXAMPLE 4

Effects of IgG on Trophoblast Annexin-V

A human trophoblast cell line (BeWo), obtained from the American TypeCulture Collection (Rockville, Md.), was maintained as described (Kohleret al., 1971, J. Clin. Endocrin. Metals. 32:683; Messmore et al., 1994,Semin. Thromb. Hemost. 20:79, both incorporated herein by reference).The BeWo cells were resuspended in a basal medium composed of a 1:1mixture of phenol red-free Ham's F12 and Dulbecco's modified Eagle'smedium plus 10 percent fetal-calf serum. They were then plated atdensities of 60,000 cells per well in 96-well culture plates and grownto confluence (approximately 130,000 cells per well). EitheraPL-antibody IgG or control IgG (2 mg per milliliter) in basal mediumplus 10 percent fetal-calf serum was added, and the cells were incubatedfor two hours at 4° C. to inhibit the recycling of membranes andvesicles. The cells were then washed once in HEPES buffer (pH 7.4)containing 5 mM calcium chloride, followed by a wash in HEPES buffercontaining 1 mM ethylene glycol—bis(β-amioethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) in place of calcium, to dissociatecell-surface annexin-V. Levels of annexin-V were determined by anenzyme-linked immunosorbent assay (ELISA) (Rand et al., 1994, Am. J.Obstet. Gynecol. 171:1566; Flaherty et al., 1990, J. Lab. Clin. Med.115:174, both incorporated herein by reference) that used a previouslycharacterized, affinity-purified, monospecific, polyclonal rabbitanti-annexin V IgG antibody (Krikun et al., 1994, Placenta 15:601,incorporated herein by reference). In assays in which known quantitiesof purified annexin-V were added, the presence of aPL or control IgG didnot in itself reduce the levels of annexin-V. All the studies wereperformed with quadruplicate culture wells. Trypan-blue exclusionstudies showed that the treated cells were at least 95 percent viable.

EXAMPLE 5

Experiments with Cultured Primary Trophoblasts

To determine whether the effects observed with the BeWo trophoblast cellline also occurred with primary cultured trophoblasts(cytotrophoblasts), the latter were obtained from women undergoingelective cesarean sections at term. The cells were isolated by amodification of the procedure of Douglas and King (J. Immunol. Meth.119:259, 1989, incorporated herein by reference) in which anti-CD45antibodies conjugated to magnetic microspheres (Advanced Magnetics,Cambridge, Mass.) were substituted for the anti-HLA antibodies used inthe original procedure (Kliman et al. 1986, Endocrinology 118:1567,incorporated herein by reference) The cells were washed, resuspended inbasal medium supplemented with 2 percent charcoal-stripped calf serumand culture supplement (ITS+, Collaborative Biomedical Products,Bedford, Mass.), and seeded in 96-well culture plates at a density of100,000 cells per well. The cultures were maintained at 37° C. in ahumidified atmosphere containing 5 percent carbon dioxide and 95 percentair, and the medium was changed at 48 hours.

The cells were allowed to form syncytia for 72 hours before the IgG wasadded, which was done in the manner described in Example 4 for thetrophoblast cell line. The final wash, with HEPES buffer containing 1 mMEGTA, was assayed for annexin-V by ELISA as described in Example 4.Since cultured primary trophoblasts do not proliferate, the results ofthese experiments were normalized for the DNA concentrations, which weredetermined by fluorimetry on the cells after their detachment, asdescribed by Hinegardner 1971, Anal. Biochem. 39:197, incorporatedherein by reference. All these experiments were performed withquadruplicate culture cells.

EXAMPLE 6

Experiments with Cultured Umbilical-Vein Endothelial Cells

Human umbilical-vein endothelial cells (HUVECs) were harvested andcultured as previously described by Jaffe et al., 1973, J. Clin. Invest.52:2757, incorporated herein by reference). They were plated at adensity of 20,000 cells per well in 96-well culture plates, allowed togrow to confluence (approximately 140,000 cells per well), and treatedin the same way as the BeWo trophoblasts. In addition to the shorttermcultures at 4° C., the HUVECs were also cultured with the IgG fractionsat 37° C. for 20 hours, after which the cells were washed once in HEPESbuffer containing 5 mM calcium chloride and then washed in HEPES buffercontaining 1 mM EGTA in place of calcium, to dissociate cell-surfaceannexin-V. The levels of annexin-V were determined by an ELISA asdescribed above (Example 4). In addition, for coagulation studies withplasma, parallel cultures of HUVECs were incubated with the IgGfractions at 37° C. for 20 hours, washed once in HEPES buffer containing5 mM calcium chloride, and then tested as described in the followingsection. Quadruplicate culture wells were used in all the studies.

EXAMPLE 7

Studies of Coagulation

After the cells were grown to confluence in the 96-well tissue-culturemicrotiter plates, studies of coagulation were performed as follows: thecells were first washed three times in HEPES buffer containing 5 mMcalcium chloride and then incubated with either antiphospholipid orcontrol IgG (5 mg per milliliter) in basal medium plus 10 percentfetal-calf serum for 90 minutes at 4° C. After a washing in HEPESbuffer, the cells were overlaid with normal pooled plasma (100 μl perwell) recalcified with 11 μl of 70 mM calcium chloride in the case ofthe BeWo cells. It was necessary to add the same volume (11 μl) of 200mM calcium chloride in order to observe coagulation of plasma in thecase of the HUVECs. Quadruplicate culture wells were used in all thestudies.

The culture plates were then placed in a kinetic microtiter-platereader, and the formation of fibrin was observed as an increase in theoptical density to 0.100 at a wavelength of 405 nm. It was confirmedthat this assay indeed monitors the formation of fibrin by determiningthat adding porcine intestinal-mucosa heparin (0.5 U per milliliter)(Steris Laboratories, Phoenix, Ariz.) or recombinant hirudin (0.5 μg permilliliter) (kindly provided by Ciba-Geigy, Summit, N.J.) to the plasmacompletely inhibited any change in optical density. Furthermore, in theabsence of heparin or hirudin the formation of fibrin nets could beobserved with the unaided eye.

In order to determine whether reducing cell-surface annexin V withoutaPL antibodies might affect the coagulation of plasma, experiments wereperformed in which HUVECs that were not incubated with human IgGfractions were washed in HEPES buffer containing 5 mM calcium chlorideand EGTA, to preserve or dissociate surface annexin-V. The HUVECs werethen incubated with rabbit polyclonal antiannexin-V IgG antibodies (100μg per milliliter) for 90 minutes at 4° C., after which they wereoverlaid with recalcified plasma and the time to coagulation measured.The controls included equivalent concentrations of polyclonal rabbitanti-annexin-II IgG (kindly provided by Dr. Katherine Hajjar, CornellUniversity Medical College) and a polyclonal rabbit antimouse idiotypeIgG (kindly provided by Dr. Thomas Moran, Mount Sinai School ofMedicine, New York, N.Y.).

In addition, HUVECs washed three times in HEPES buffer that contained 5mM. calcium chloride, to preserve cell-surface annexin-V, were comparedwith cells that were washed three times in HEPES buffer containing 1 mMEGTA, to dissociate cell-surface annexin-V. Each of these treatments wasfollowed by a washing in buffer containing calcium chloride, after whichthe cells were overlaid with recalcified normal pooled plasma containingvarious concentrations of annexin-V; the HUVECs were then monitored forcoagulation as described above. In addition, the coagulation times ofthe HUVECs incubated with plasma containing recombinant annexing at aconcentration of 4 μg per millileter (kindly provided by Dr. Hajjar)were compared with those of cells incubated with plasma containingannexin-V in the same concentration and cells incubated withHEPES-buffer control.

Statistical Analysis

All the statistical analyses herein were performed with the use ofStudent's two-tailed t-test (InStat program, Graphpad, San Diego,Calif.).

EXAMPLE 8

Effects of Antiphospholipid Antibodies on Annexin-V and PlasmaCoagulation in Trophoblasts

The effects of aPL IgG on levels of annexin-V associated with thetrophoblast cell surface, using the BeWo trophoblast cell line (Example4) was studied. With each of the three different aPL IgG antibodies(Example 1), the amount of annexinV associated with the trophoblast cellsurface was significantly lower than that associated with control IgG,and the reductions were similar (FIGS. 1A and 1B). It was thendetermined whether these reductions also occurred with primary culturedplacental trophoblasts (cytotrophoblasts) (Example 5). When thesetrophoblasts were incubated with aPL IgG, there was a significantlylower amount of annexin-V, approximately 20 percent of the amount foundin trophoblasts incubated with control IgG (FIG. 1B).

It was then tested whether the reduction in the amount of the annexin-Vanticoagulant protein was associated with a shortening in thecoagulation time of a plasma exposed to these cells. There was indeed asignificant shortening in the clotting times of plasma on thetrophoblasts exposed to antiphospholipid IgG, as compared with thoseexposed to control IgG (FIG. 1C).

EXAMPLE 9

Effects of Antiphospholipid Antibodies on Annexin-V and Plasma

Coagulation in Umbilical-Vein Endothelial Cells The aPL-antibodysyndrome may lead to thrombosis in veins and arteries. In view of thefindings with trophoblasts (Example 8), the effects of aPL antibodies onlevels of annexin-V and plasma coagulation on the surfaces of HUVECs. Aswas found with trophoblasts (Example 8), levels of annexin-V werereduced on the surface of epithelial cells exposed to antiphospholipidantibody (FIG. 2A). There was also a significant acceleration ofcoagulation on the surface of HUVECs exposed to aPL IgG as compared withcontrol IgG (FIG. 2B). The results were similar with HUVECs cultured at37° C. for 20 hours with the antibodies (FIGS. 2C and 2D).

When HUVECs not treated with aPL IgG were incubated with rabbitpolyclonal anti-annexin-V IgG, the coagulation time of plasma applied tothe cells was significantly shorter than after incubation with antimouseIgG, and treating the HUVECs with anti-annexin II IgG had no effect onthe coagulation time (FIG. 3A). This shorter coagulation time did notoccur with cells from which the annexin-V was first dissociated withEGTA (FIG. 3A). Also, removing annexin-V from the endothelial surface bypreincubation with EGTA significantly reduced the coagulation time(FIGS. 3A and 3B). Furthermore, adding exogenous annexin-V resulted indose-dependent prolongations of coagulation in both cells whoseannexin-V had been removed by EGTA treatment and controls whoseannexin-V had been preserved by treatment with calcium-containing buffer(FIG. 3B). In contrast, there was no difference in the mean (±SE)coagulation time between the epithelial cells exposed to plasmacontaining 4 μg of annexin II pre milliliter and the controls exposed tobuffer alone (19.5±0.6 vs. 19.8±0.2 minutes).

EXAMPLE 10

Ellipsometry Studies

The effects of aPL antibodies on phospholipid-bound annexin-V werestudied using computer-assisted ellipsometry (Andree et al. 1992, inAndree (ed.) Phopholipid Binding and Anticoagulant Action of Annexin-V,Maastricht, the Netherlands, Universitaire per Maastricht, p.73,incorporated herein by reference). Planar phospholipid bilayers wereapplied to silicon slides as previously described. (Andree et al., 1992,J. Biol. Chem. 267:17907; Giesen et al., 1991, J. Biol. Chem. 266:1379,both incorporated herein by reference.) A 5 mM vesicle mixture of 30%1,2-dioleoyl-snglycero-3-phosphatidyl serine and 70%1,2-dioleoyl-sn-glycero-3-phosphatidyl choline (PS/PC) (AvantiPolar-Lipids, INC. Alabaster, Ala.) was dried under nitrogen andsonicated (Sonic Dismembrator model F60, Fisher Scientific, Pittsburgh,Pa.) in HEPES buffer (0.01 M HEPES, 0.14 M NaCl, pH 7.5) at 0° C. untilthe suspension was completely clear. Silicon slides of 1 by 4 cm and 0.4mm thickness were cut from silicon wafers (Wacker Chemie, Munich,Germany, n-type, phosphor-doped). The slides were thoroughly cleanedwith detergent (Sparkleen, Fisher Scientific Company, Pittsburgh, Pa.)and water. Then they were kept overnight in 30% chromic sulfuric acid,flushed with water and stored in 50% alcohol-water until use, when theywere rinsed with distilled water and then dipped into a containercontaining stirring HEPES buffer composed of 0.01 M HEPES, 0.14 MNaCl,0.1% BSA, 5 mM CaCl₂, pH 7.5. The sonicated PS/PC vesicles (finalconcentration 50 μM) were then added and stirred for 10 min. Each slidewas flushed with the latter HEPES buffer and transferred to anellipsometer cuvette containing the stirring buffer. Adsorption ofannexin-V (2 μg/ml), β₂-glycoprotein I(β₂-GP I) (2 μg/ml, provided byDr. K. McCrae, Temple University) and IgG preparations (100 μg/ml) tothe phospholipid bilayers on the silicon slide were observed after theserial addition of each of the proteins. The mass of the PS/PC bilayer(˜0.4 μg/cm²) was measured and subtracted for these curves. Afteradsorption had reached equilibrium, residual annexin-V was desorbed fromthe phospholipid bilayers on the silicon slide by the addition of 0.5 MEDTA (to a final concentration of 6 mM) and measured. Addition of EDTAhad no effect upon the adsorptions of IgGs or β₂-GP I-, alone or incombination. The completeness of desorption of annexin-V from thesurface by EDTA was further confirmed by subsequently solubilizing thephospholipid bilayers with 0.1% SDS. The SDS solubilized samples werechecked for annexin-V by standard immunoblot with a monospecificpolyclonal rabbit anti-annexin-V IgG following 0.1% SDS-12%polyacrylamide gel electrophoresis as described in Krikun et al., 1994,Placenta 15:601, incorporated herein by reference.

EXAMPLE 11

Annexin-V Binding to Phosphatidyl Serine-Coated Microtiter Plates

Microtiter plates (Nunc-Immuno Plate, MaxiSorp Surface) (FisherScientific, Pittsburgh, Pa.) were coated with phosphatidyl serine PS(Avanti Polar-Lipids, INC. Alabaster, Ala.) as previously described(Yamamoto et al., 1990, Clin. Exp. Immunol. 94:196; Rote et al., 1990,Am J. Obste.t Gynecol. 163: 575, both incorporated herein by reference,)and used as the phospholipid substrate. Citrated plasma samples (50 μl)were added to each well in duplicate and incubated for 30 minutes atroom temperature. The wells were emptied and washed 4× with PBS buffer,pH 7.4. 50 μl of biotinyiated-annexin-V (1 μg/ml in TBS buffercontaining 0.1% BSA and 5mM CaCl₂, pH 7.4) was then added to each welland incubated for 30 minutes at room temperature. The wells were washed4× with PBS buffer, followed by 50 μl of phosphatase-labeledstreptavidin (0.5 μg/ml in TBS buffer containing 0.1% BSA) (Kirkegaard &Perry, Laboratories, Gaithersburg, Md.), which was incubated for 30minutes at room temperature. The wells were then emptied and washed 4×with PBS buffer, pH 7.4. 50 μl of p-nitrophenyl phosphate substrate (1mg/ml in DEA buffer) (Sigma Chemical Company, St. Louis, Mo.) were addedand incubated for approximately 30 minutes at room temperature, afterwhich the optical absorbance was read at 405 nm with a kineticmicroplate reader (Molecular Devices, Menlo Park, Calif.).

EXAMPLE 12

Annexin-V Binding/Desorption Assays and Coagulation Studies with WashedPlatelets

Platelets were prepared for coagulation studies according to the methodpreviously described for the platelet aPTT test (“plateletneutralization procedure”) (Thiagarajan et al., 1990, J. Biol. Chem.265:17420, incorporated herein by reference). Pooled platelets fromblood bank donors were washed 3× in TBS buffer (0.15 M NaCl, 0.02 MTris) containing I mg/ml glucose, pH 7.4, resuspended to a density of2×105/μl in the buffer, aliquotted and stored at −70° C. for allstudies.

The effects of aPL IgGs on the quantity of platelet-associated annexin-Vbound by platelets were performed with modified methods similar to thosedescribed above for cultured trophoblasts and endothelial cells.(Examples 4 and 6). aPL and control IgGs were added to normal citratedplasma to a fmal concentration of 5 mg/ml. Aliquots of frozen and thawedwashed platelets (1×10⁸) were incubated with 100 μl of the plasmas at 4°C. for 2 hrs, as described above. The platelets were washed 3× andresuspended in HEPES buffer containing 5 mM CaCl₂, pH 7.4. Annexin-V wasthen added to the platelets in the HEPES buffer containing 5 mM CaCl₂ toa final concentration of 20 μg/ml (determined after pilot studies withvarying concentrations of annexin-V) and incubated at 4° C. for anadditional 15 minutes. The platelets were then centrifuged and washed 3×in the same buffer. After the final centrifugation, to dissociateannexin-V, the platelets were resuspended in the HEPES buffer containingI mM EGTA. The quantity of platelet associated annexin-V was determinedby an ELISA (as described in Examples 4 and 6). The results of theassays were expressed as ng of annexin-V/10⁶ platelets.

The effects of aPL IgG on coagulation using platelets exposed to aPL orcontrol IgGs were also studied. For these experiments the IgGs wereadded to pooled normal plasma to a concentration of 5 μg/ml. Thawedwashed platelets (120 μl), at a density of 2×10⁵/μl in the TBS bufferdescribed above, were added to 120 μl of IgG-containing plasma andpre-incubated for 10 min at 37° C. The platelets were centrifuged,washed 2× and resuspended in 120 μl of the TBS buffer. A 50 μl volume ofthe platelet suspension was then added to 50 μl of pooled normal plasma,incubated for 30 sec at 37° C. in a ST4 Coagulation Instrument (AmericanBioproducts Co. Parsipanny, N.J.). 50 μl of Celite (5 g/L in TBS bufferconsisting of 0.05 M Tris, 0.1 M NaCl, pH 7.4) was added and the mixturewas incubated for 60 sec. A volume of 50 μl of 0.02 M CaCl₂, or 0.02 MCaCl₂ containing annexin-V at 20 μg/ml was then added, the times untilclot formation were measured and the mean times of duplicate tests werereported. Coagulation times of specimens without and with annexin-V wererecorded, along with the net prolongations (ie. coagulation time in thepresence of annexin-V minus coagulation time in the absence ofannexin-V) which reflected the anticoagulant activity of the annexin-V.

EXAMPLE 13

Annexin-V Binding/Desorption Assays and Coagulaton Studies with aPTTReagent

The effects of aPL IgG on the quantity of annexin-V associated with apartial thromboplastin time (aPTT) reagent-phospholipid (Actin FS)(Baxter Diagnostics Inc., Deerfield, Ill.) was studied. 100 μl of theaPTT phospholipid-reagent was incubated with 100 μl of aPL patientplasma or control plasma for 15 minutes at 37° C. The mixture of aPTTreagent-phospholipid and plasma was sedimented with a microcentrifuge(Model 5451, Brinkmann Instruments, Inc, Westbury, N.Y.) at 15,000×g for20 minutes at room temperature. The plasma treated-phospholipid pelletswere washed 3× with HEPES buffer (0.01 M HEPES, 0.14 M NaCl, 0.1% BSA,pH 7.4) and resuspended in HEPES buffer containing 5 mM CaCl₂. Annexin-Vwas then added into the phospholipid suspension to a final concentrationof 2 μg/ml and incubated at 37° C. for 10 minutes. After centrifugation,the phospholipid pellets were washed 3× in HEPES buffer containing 5 mMCaCl₂. The phospholipid-associated annexin-V was then dissociated fromthe aPTT phospholipid-reagent with HEPES buffer containing 5 mM EDTA andassayed by ELISA as described above. The results of the assays wereexpressed as ng of annexin-V/50 μl aliquot of aPTT phospholipid-reagent.

The effect of aPL IgG on coagulation with aPTT reagent-phospholipid wasdetermined using a 2 stage assay, in which the first stage exposed aPTTsreagentphospholipid to potential aPL antibodies in the test plasma, andthe second stage measured coagulation times with the phospholipid usinga pooled normal plasma. This assay was designed using the pooled normalplasma for the second stage in order to deal with the variable baselinecoagulation times of individual patients. 50 μl of the aPTTreagent-phospholipid was added to 50 μl of aPL patient or controlcitrated plasma. The mixture was sedimented with the microcentrifuge, asdescribed above. The pellets were then washed once in the TBS buffer andresuspended in 220 μl of this buffer. 50 μl of this plasma treatedphospholipid suspension was added to 50 μl pooled normal plasma andincubated for 120 sec at 37° C. in the ST4 Coagulation Instrument, afterwhich 50 μl of 0.02 M CaCl₂ or 0.02 M CaCl₂ containing annexin-V (30μg/ml) was added. The times until clot formation of duplicate specimenswere monitored and reported, as described above for platelets (Example12).

EXAMPLE 14

Annexin-V Binding/Desorption Assays and Coagulation Studies withProthrombin Time Reagent

The effect of aPL IgG on the quantity of annexin-V associated withprothrombin time (PT) reagent (tissue factor) was studied. PT reagent(ThromboplastinC Plus, Baxter Diagnostics Inc, Deerfield, Ill.) waswashed 3× with TBS buffer by sedimentaion and resuspension with themicrocentrifuge for 20 minutes at 15,000×g, as described for the aPTTphospholipid-reagent (Example 13). 100 μl of the washed phospholipid ofPT reagent was incubated with 100 μl of aPL or control IgG-containingplasma (at a concentration of 5 mg/ml, prepared as for the plateletsdescribed above in Example 12) for 15 minutes at 37° C. The mixture wasthen centrifuged and the pellets of IgG-treated PT reagent-phospholipidwere then treated in the same way as described above for aPTTreagent-phospholipid (Example 13). The phospholipid-associated annexin-Vwas dissociated from the PT phospholipid-reagent by the HEPES buffercontaining 5 mM EDTA the and was assayed by ELISA, as described above.The results were reported as described for the aPTT reagent embodimentof this method, above.

In order to investigate the effects of aPL IgG on coagulation with PTreagent, a two-stage assay, similar to the aPTT assay described above(Example 13), was used. The PT reagent (50 μl) was washed 3× in TBSbuffer to remove calcium, and was added to 50 μl of aPL patient orcontrol plasma. The mixtures were centrifuged as described above and thepellets were washed once in the TBS buffer and resuspended in 220 μl ofthis buffer. 50 μl of the suspension was then added to 50 μl poolednormal plasma and incubated for 120 sec at 37° C. in the ST4 CoagulationInstrument. A 50 μl volume of 0.02 M CaCl₂ or 0.02 M CaCl₂ containingannexin-V (30 μg/ml) was added. The times until clot formation ofduplicate specimens were monitored and recorded as described above.

EXAMPLE 15

Fluorometric Determination of Annexin-V Binding to Phospholipid

The effect of aPL plasma on the binding of fluorescein isothiocyanate(FITC)-conjugated annexin-V to phospholipid was also determined. 50 μlof aPTT phospholipid-reagent was added to 50 μl of aPL or control plasmaand sedimented in the microcentrifuge as described above. The pellets ofplasma treated-phospholipid were washed once in the TBS buffer, pH 7.4,and then resuspended in 55 μl of the above buffer containing 5 mM CaCl₂and 1 μg/ml of FITC-conjugated annexin-V (Clontech Laboratories, Inc.Palo Alto, CA). The mixture was incubated for 5 minutes at roomtemperature and centrifuged as described above, after which thesupernatants were collected and the pellets were resuspended in 55 μl ofthe TBS buffer containing 10 mM EDTA. The levels of labeled annexin-V inthe supernatant and bound to the aPTT phospholipid-reagent were measuredin terms of relative fluorescence intensity with an SLM/Amico SPF-500Cspectrofluorometer (Milton Co. Rochester, N.Y.). The samples werecontained in 50 μl quartz microcuvettes, thermostated at 20° C.Excitation was at 490 nm with a 4-nm band-pass and emission was detectedat 523 nm with a 20-nm band-pass. The results were reported as relativefluorescence units (RFU). A standard curve of fluorescence intensityversus FITC-annexin-V concentration was linear between 0 and 2 μg/ml ofprotein.

EXAMPLE 16

Results of Ellipsometry Studies

The quantity of annexin-V adsorbed to PS/PC (30%/70%) phospholipidbilayers on silicon slides was determined as described in Example 10,and the amount of annexin-V remaining on the phospholipid bilayers afteraddition of IgG preparations was determined by adding EDTA to desorb theresidual annexin-V. All of the annexin-V which bound to the phospholipidsurface was subsequently desorbed by addition of 6 mM EDTA (FIG. 5A).Since the adsorptions of IgGs and β₂-GP I were not affected by EDTAtreatment, EDTA-induced desorption could be used to measure the amountof annexin-V remaining on the phospholipid surface after addition of aPLIgG antibodies. Measurement of the binding of 3 pairs of aPL IgG fromaPL patients (Example 1) to the planar phospholipid bilayers ofPS/PC-bound annexin-V showed that aPL IgG binding to the phospholipidbilayers, in the presence of (B₂-GP I cofactor, displaced 0.115±0.014μg/cm² (mean±SEM) annexin-V from the phospholipid surface (FIGS. 5C &6). In contrast, control IgG with the cofactor did not displaceannexin-V (0.005±0.007 μg/cm², p=0.002) (FIGS. 5D & 6). Also, aPL IgGwithout the cofactor did not reduce the amount of adsorbed annexin-V(FIG. 5B), indicating that the cofactor was required when aPL IgGs wereused. Immunoblotting of SDS solubilized slides after the EDTA desorptionshowed no residual annexin-V (data not shown).

EXAMPLE 17

Results of Annexin-V Binding to Phosphatidyl Serine-Coated MicrotiterPlates

PS-coated microtiter plates treated with aPL plasmas (Example 11) boundsignificantly less biotin-labeled annexin-V than PS-coated microtiterplates which had been treated with control plasmas (FIG. 7). The mean OD(+SEM) of the aPL plasmatreated wells was 0.085±0.003 compared to0.123±0.005 for the wells teated with control plasmas (n=10 for eachgroup, p<0.0001).

EXAMPLE 18

Results of Platelet Experiments

The frozen thawed washed platelets (Example 12) which had been incubatedwith annexin-V after preincubation with aPL IgG preparations from the 3different patients had significantly less annexin-V bound to theirsurfaces (mean±SEM-. 0.89±0.12 ng/10⁶ platelets) than platelets whichhad been pre-exposed to control IgGs (2.01±0.38 ng/10⁶ platelets,p=0.05, n=3) (FIG. 8A). Although annexin-V increased the coagulationtimes with both aPL and control IgG treatments, the protein had less ofan anticoagulant effect with aPL IgG-treated platelets—i.e., there wassignificantly less prolongation with thawed washed platelets which hadbeen pre-incubated with aPL IgG (mean±SEM−33.2±0.9 sec longer thancoagulation time in absence of annexin-V) compared to control IgG(50.4±4.1 sec longer than coagulation time in absence of annexin-V,p=0.01, n=3) (FIG. 8B).

EXAMPLE 19

aPTT Reagent Results

In experiments utilizing aPTT reagent-phospholipid (Example 13), theaPTT reagent-phospholipid pre-exposed to aPL plasma was found to bindsignificantly less annexin-V (mean±SEM−318±28 ng/50 μl aliquot ofreagent) than controls (656±80 ng/50 μl aliquot of reagent, p=0.01, n=4)(FIG. 9A). A two-stage test was designed to measure coagulation, inwhich aPTT reagent-phospholipid was first incubated with individual testplasma, washed, and for the second stage, exposed to a pooled normalplasma, which was then recalcified (to allow for caogulation to occur)in the presence and absence of added annexin-V. It was found thatpre-exposure of aPTT reagent-phospholipid to plasmas from aPL syndromepatients significantly accelerated the coagulation of pooled normalplasma in the presence of annexin-V (mean±SEM:89.2±2.2 sec) as comparedto control plasmas (mean+SEM−102.5±2.6 sec, p=0.001, n=10) (FIG. 9B).Also, there was a commensurate decrease in the annexin-V-inducedanticoagulant effect, as assessed by prolongation of the coagulationtime, of the aPL treated reagent as compared to the reagent which hadbeen pre-incubated with control plasma (mean±SEM−13.6±1.8 sec for aPLpatients and 23.1±0.8 sec for controls, p=0.0002) (FIG. 9B).

EXAMPLE 20

Prothrombin Time Reagent (tissue factor-phospholipid) Results

Similar experiments with tissue factor-phospholipid suspensions (PTreagent) (Example 14) also showed significantly less binding ofannexin-V to PT reagentphospholipid which had been pre-exposed to aPLIgG fractions (mean±SEM−82±4 ng/50 μl aliquot of reagent) than controls(110±1 ng/50 μl aliquot of reagent, p=0.02, n=3) (FIG. 10A). In thepresence of annexin-V, PT reagent which had been pre-exposed to aPLplasmas accelerated the subsequent coagulation of pooled normal plasma(mean±SEM:35.0±0.8 sec) as compared to controls (38.3±1.2 sec, p=0.03,n=10) (FIG. 10B). There was a corresponding decrease in theannexin-V-induced anticoagulant effect with PT reagent which had beenpre-incubated with aPL plasma (mean±SEM−0.10.3±0.8 sec) as compared tocontrols (15.2±1.2 sec, p=0.004). In contrast, in the absence ofannexin-V, aPL-treated prothrombin time reagent caused a small butsignificant slowing of coagulation compared to prothrombin time reagentwhich had been pre-incubated with control plasma (mean±SEM:24.7±0.5 secfor aPL-treated reagent compared to 23.1±0.1 sec for controls, p=0.003)(FIG. 10B).

EXAMPLE 21

FITC-Annexin-V Binding to Phospholipid

Pre-exposure of aPTT reagent-phospholipid to aPL plasma was found tosignificantly reduce the amount of FITC-conjugated annexin-V (Example15) which subsequently bound to the phospholipid (mean±SEM−0.083±0.008RFU) as compared to control plasmas (0.131±0.015 RFU, p=0.01, n=10)(FIG. 11), and increase the amount of labeled annexin-V in thesupernatant (mean+SEM−0.070±0.008 RFU for aPL exposed phospholipid and0.046±0.005 RFU for control plasmas, p=0.02) (FIG. 11).

EXAMPLE 22

Effects of Antiphospholipid Antibodies and Annexin-V on Prothrombinase

The effect of annexin-V and aPL antibodies on phospholipid dependentprothrombinase (Factor Xa) was assayed using the following methodology.PS/PC (30%/70%, 5 mM, size 100 nanometers in diameter) vesicalsuspension was made by filtration through 100 nanometer membrane filterswith an extruder, in a manner similar to that described in Example 10.Phospholipid bilayer coated slides were made as described in Example 10.The slides were transferred to an ellipsometer cuvette containing HEPESbuffer (0.01 M HEPES, 0.14 M NaCl, pH 7.5) containing 1.25 mM CaCl₂,0.02% BSA. Protein adsorption by the slides was observed with theellipsometer. β₂-GP I (3 μg/ml) and aPL IgG (0.5 mg/ml) was then addedto the cuvette, the proteins were absorbed at room temperature for 15minutes. After adsorption had reached equilibrium, annexin-V was thenadded to a final concentration of 7 μg/ml. The adsorption reachedequilibrium in 15-20 minutes. After which the slide was flushed with 10ml of HEPES buffer containing 1.25 mM CaCl₂, 0.02% BSA, followed by 10ml of HEPES buffer containing 5 mM CaCl₂, 0.1% BSA. Factor Xa (100 pM)and factor Va (1 nM) were then added. After adsorption for 10 minutes,prothrombin was added to a final concentration of 25 nM, sampling wasdone at intervals every 1 minute. 50 μl of sample was added intomicrotiter plate well which had been pre-filled with 100 μl of 36 mMEDTA in bicine buffer. 25 μl of the chromogenic substrate for thrombin,S2238 (Chromogenix, Sweden), was added to each well. Thrombin generationwas monitored with a kinetic reader at wavelengths of 405 nm and 490 nm.

Annexin-V suppressed the phospholipid dependent activity ofprothrombinase, and factor Xa, consequently the formation of thrombin.The addition of aPL antibodies and cofactor β₂-GP I to this reaction,resulted in the observation that annexin-V no longer suppressed theformation of thrombin. Whereas, control (non-aPL) antibodies/β₂-GP I hadno effect on the formation of thrombin.

All references not previously specifically incorporated herein byreference are hereby incorporated herein.

I claim:
 1. A method of diagnosing and/or monitoring antiphospholipidantibody syndrome in a patient comprising a. incubating a phospholipidsubstrate with a test blood specimen suspected of containingantiphospholipid antibody from the patient in the presence of a knownamount of an annexin for a time sufficient to allow the annexin to bindto the phospholipid substrate, b. removing unbound test specimen andannexin from the phospholipid substrate, c. measuring the amount ofannexin bound to the phospholipid substrate in the presence of the testspecimen, and d. comparing the amount of annexin bound in the presenceof the test specimen to the same known amount of annexin bound to aphospholipid substrate in the presence of a control blood specimen,wherein a lower amount of annexin bound to the phospholipid substrate inthe presence of the test specimen compared with the control specimenindicates the presence of antiphospholipid antibody syndrome in theindividual.
 2. A method of diagnosing and/or monitoring aantiphospholipid antibody syndrome in a patient comprising a. incubatinga phospholipid substrate with a test blood specimen suspected ofcontaining antiphospholipid antibody from the patient in the presence ofa known amount of an annexin for a time sufficient to allow the annexinto bind to the phospholipid substrate, b. removing unbound test specimenand annexin from the phospholipid substrate, c. measuring the amount ofunbound annexin from step b, and d. comparing the amount of unboundannexin from step b to an amount of unbound annexin in a sample ofannexin bound to a phospholipid substrate in the presence of a controlblood specimen, wherein a higher amount of unbound annexin in thepresence of the test specimen compared to the amount of unbound annexinin the presence of the control specimen indicates the presence ofantiphospholipid antibody syndrome.
 3. The method of claim 1 or 2wherein the annexin is annexin-V.
 4. The method of claim 1 or 2 whereinthe test specimen and control specimen comprise anticoagulated plasma.5. The method of claim 1 or 2 wherein the test specimen and controlspecimen comprise isolated IgG.
 6. The method of claim 5 wherein theincubation is carried out in the presence of a cofactor that enhancesantiphospholipid antibody binding to the phospholipid substrate.
 7. Themethod of claim 6 wherein the cofactor is β₂-glycoprotein I.
 8. Themethod of claims 1 or 2 wherein the phospholipid substrate is selectedfrom among cultured trophoblasts, endothelial cells, other cells andcultured cell lines having surface anionic phospholipids, platelets,phospholipid coated silicon wafers, phospholipid coated microtiterplates, phospholipid coated beads, phospholipid suspensions andcoagulation test reagents comprising phospholipids.
 9. The method ofclaim 8 wherein the coagulation test reagent is selected from amongpartial thromboplastin time reagent and prothrombin reagent.
 10. Themethod of claim 8 wherein the phospholipid substrate comprises aphospholipid selected from among phosphatidyl serine, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin,phosphatidic acid and combinations thereof.
 11. The method of claim 8wherein the phospholipid substrate is a phospholipid coated microtiterplate or phospholipid coated beads.
 12. The method of claim 8 whereinthe phospholipid substrate is platelets.
 13. The method of claim 8wherein the phospholipid substrate is partial thromboplastin timereagent.
 14. The method of claim 8 wherein the phospholipid substrate isprothrombin reagent.
 15. The method of claim 1 or 2 wherein the annexincomprises a detectable label.
 16. The method of claim 15 wherein thedetectable label is selected from among biotin, a fluorochrome, aradioisotope, a chemiluminescent label and a chromophore.
 17. The methodof claim 16 wherein the fluorochrome is fluorescein isothiocyanate. 18.The method of claim 1 further comprising removing bound annexin from thephospholipid substrate.
 19. The method of claim 18 wherein the annexinis removed by desorbing with a calcium chelating agent.
 20. The methodof claim 19 wherein the calcium chelating agent is EDTA or EGTA.
 21. Themethod of claim 1 wherein bound annexin is measured by an ELISA,streptavidin binding, fluorescence or chemiluminescence.
 22. The methodof claim 19 wherein desorbed annexin is measured by an ELISA,streptavidin binding, fluorescence or chemiluminescence.